Poly(ADP-ribose) synthesis: a useful parameter for identifying apoptotic cells.
Cell death by apoptosis was analysed in HeLa cells either treated with the antitumoral drug bleomycin or depleted of growth factors by long-term culture without medium change. The interference of apoptosis with normal cell cycle progression was followed by flow cytometry in cells stained with propidium iodide and with antibody to S-phase-related PCNA protein. Bleomycin-treated cells showed a net accumulation in G2/M phase paralleled by the appearance of material with a subdiploid DNA content. Cells with a subdiploid DNA content were also present in growth factor-depleted cultures and were shown to derive from all the cell cycle phases. To identify apoptotic features in HeLa cell cultures, we applied a recently developed assay based on the simultaneous analysis in the single cell of three parameters, namely chromatin condensation, DNA degradation and poly(ADP-ribose) synthesis. Apoptotic cells were visualized by sequential reactions: Hoechst staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and immunoreaction with anti-poly(ADP-ribose) monoclonal antibody. Positive reactions were obtained for cells at different stages of the apoptotic programme showing condensed nuclei, fragmented chromatin and apoptotic bodies.
Donzelli
M
M
Istituto di Genetica Biochimica Evoluzionistica CNR, Pavia, Italy.
Negri
C
C
Mandarino
A
A
Rossi
L
L
Prosperi
E
E
Frouin
I
I
Bernardi
R
R
Bürkle
A
A
Scovassi
A I
AI
D017209:Apoptosis / Q000502:physiology*; D006690:Bisbenzimidazole; D001761:Bleomycin / Q000494:pharmacology; D002453:Cell Cycle; D002843:Chromatin / Q000378:metabolism; D003470:Culture Media; D053938:DNA Fragmentation; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis*; D011419:Propidium
D002843:Chromatin; D003470:Culture Media; D001761:Bleomycin; D011064:Poly Adenosine Diphosphate Ribose; D011419:Propidium; D006690:Bisbenzimidazole